Bactericidal and virucidal pharmaceutical composition

ABSTRACT

A bactericidal and virucidal pharmaceutical composition for use on epithelial tissues such as pulmonary, nasal and oral tissues, which comprises a non-steroidal anti-inflammatory drug (NSAID) in a concentration between 5 and 500 mM and a salt, being the NSAID preferably solubilized in a hypertonic saline solution applicable in therapies for viral infections of the Herpes simplex type. The composition can be used in therapies for herpes simplex viral infections, be used as a bactericidal mouthwash, or be vehiculated to the lung by using a nebuliser, for cystic fibrosis.

RELATED APPLICATIONS

This application is the US national phase application of internationalapplication number PCT/ES2016/070702, filed 4 Oct. 2016, whichdesignates the US and claims priority to Argentinian application AR20150103203 filed 5 Oct. 2015, the contents of each of which are herebyincorporated by reference as if set forth in their entireties.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions for thetreatment of pulmonary diseases and of epithelial tissue, such as cysticfibrosis. In particular, this invention is a composition meant for topicuse or nebulisations.

STATE OF THE ART

Non-steroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous groupof drugs which share their therapeutical action (analgesic,anti-inflammatory and antipyretic effect) but differ in their relativetoxicity and efficacy.

Although NSAIDs are relatively safe are drugs, when administered insuitable doses and to selected patients they may present potentiallysevere adverse effects and interactions. The side effects caused byNSAIDs affect various organs, but those generated at thegastrointestinal level are the most frequent ones. About 2-3% ofpatients taking NSAIDs for a year develop gastrointestinal complicationssuch as bleeding, or upper or lower perforation. Over that period, 5-10%desarrollan úlceras sintomáticas y un 30-50% desarrollan dispepsia querequiere atenćion {acute over (m)}edica [Lanas, A., Pique, J. M., Ponce,J., Gastroenterol. Hepatol., 24, 22 (2001); Gastroenterol. Hepatol., 24,134 (2001). 2].

An ibuprofen molecule with a MW 206.3 g/mol, (FIG. 1) as well as otherderivatives of 2-arylpropionate, which include ketoprofen, flurbiprofen,naproxen, etc., contains a chiral carbon in the α (alpha) position ofthe propionate.

Ibuprofen is considered a non-steroidal anti-inflammatory agent (NSAID)frequently used as antipyretic and for the symptomatic relief ofheadache (cluster), dental pain, muscle pain or myalgia, menstrualdiscomfort, mild neurological pain, and post-surgical pain. It is alsoused to treat inflammatory conditions such as those present inarthritis, rheumatoid arthritis and gouty arthritis. Generally, therecommended adult dose is about 1200 mg daily. However, under medicalsupervision, the maximum amount of ibuprofen for adults is 800 mg perdose or 3200 mg per day.

There is little correlation between the severity of symptoms and theplasma levels measured. Toxic effects are unlikely to appear with dosesbelow 100 mg/kg but they can be severe above 400 mg/kg (about 150tablets of 200 mg for a normal patient). However, high doses do notindicate that the clinical features are to be lethal. It is not possibleto determine a precise lethal dose, since it can vary with age, weightand associated disease in patients.

Most symptoms are an excess of action and include abdominal pain,nausea, vomiting, dizziness, drowsiness, headache, tinnitus andnystagmus. Symptoms may be rarely more severe, being some of themgastrointestinal bleeding, seizures, metabolic acidosis, hyperkalemia,hypotension, bradycardia, tachycardia, atrial fibrillation, coma, liverfailure, acute renal failure, cyanosis, respiratory depression andcardiac arrest.

Ibuprofen, in structure, is a substantially water-insoluble molecule. Itsolubilizes less than 1 mg in 1 mL of water (<1 mg/mL). The form ofadministration of ibuprofen for the treatment of all the cases mentionedabove is always in tablets or soft capsules in which ibuprofen is foundin protonated form. Therefore, such protonated form renders ibuprofen amarkedly water-insoluble molecule. As a counterpart, ibuprofen issoluble in organic solvents such as ethanol or acetone.

Ibuprofen may be solubilised in water by removing the proton by somecation. In this regard, an aqueous solution up to concentrations of 1 MpH 7.0-8.5, titrating ibuprofen with alkaline solutions of sodiumhydroxide, potassium hydroxide, magnesium hydroxide may be prepared, orelse diethanolamine, tris (hydroxymethyl) aminomethane (THAM) orarginine, lysine or histidine may be added.

Because of its molecular structure, once solubilised in water anddepending on its concentration, ibuprofen becomes an amphipathicmolecule with surfactant properties with two forms or structures, onebelow its critical micelle concentration (CMC) that is about 180 mM(Amphiphilic association of ibuprofen and two nonionic cellulosederivatives in aqueous solution—Annika Ridell, Hans Evertsson, StefanNilsson and Lars-Olof Sundeof—Journal of Pharmaceutical Sciences. Volume88, Issue 11, pages 1175-1181—November 1999) and another over its CMC,where it is found forming a micelle.

In vitro studies indicate that, like other NSAIDs, ibuprofen is largelybound to plasma albumin, although in babies, this appears to besignificantly lower (95%) compared with adult plasma (99%). Ibuprofencompetes with bilirubin binding to albumin. Especially in the serum ofnewborn babies, this could result in the increased free fraction ofbilirubin at high concentrations of ibuprofen.

Hussein and Janabi have recently published an article (In VitroAntibacterial Activity of ibuprofen and Acetaminophen, J Glob InfectDis. 2010 May-August; 2(2): 105-108), in which in vitro antibacterialactivity of ibuprofen and acetaminophen is discussed. The articlementions that ibuprofen limits its bactericidal activity only tobacteria such as Escherichia coli (Celik I, Akbulut A, Kilic S S, RahmanA, Vural P, Canbaz M, Felek S. Effects of ibuprofen on the physiologyand outcome of rabbit endotoxic shock. BMC infectious diseases. 2002;2:26-38. Bernard G R, Wheeler A P, Russell J A, Schein R, Summer W R,Steinberg K P, et al. The effects of ibuprofen on the physiology andsurvival of patients with sepsis. New Engl J Med. 1997. In this case itis proved that ibuprofen presents its anti-inflammatory activity on thelungs of rabbits infected with Pseudomonas aeruginosa, but does not haveeffects on the bacteria itself (Sordelli D O, Cerquetti M C, el-Tawil G,Ramwell P W, Hooke A M, Bellanti J A. Ibuprofen modifies theinflammatory response of the murine lung to P. aeruginosa. Eur J RespirDis. 198567118-27. Such work also shows that ibuprofen does not presentactivity on ampylobacter pylori in humans (Graham D Y, Klein P D, OpekunA R, Smith K E, Polasani R R, Evans D J, et al. In vivo susceptibilityof Campylobacter pylori. Am J Gastroenterol. 1989; 84:233-8] y sobreMycobacterium tuberculosis in rats (Byrne S T, Denkin S M, Zhang Y.Aspirin and ibuprofen enhance pyrazinamide treatment of murinetuberculosis. J Antimicrob Chemoth. 2007; 59:313-6). In this study asignificant activity of ibuprofen is not shown since no effect wasobserved on bacteria type Serratia or Bacillus subtilis, and the authorspresent a varying inhibitory activity of ibuprofen. It is worthmentioning that the in vitro studies were conducted vehiculizingibuprofen in ethanol, a condition that causes precipitation of ibuprofenitself when the stock solution is diluted in aqueous medium.

The research work developed by Sordielli et al. (Sordelli D O, CerquettiM C, el-Tawil G, Ramwell P W, Hooke A M, Bellanti J A. ibuprofenmodifies the inflammatory response of the murine lung to Pseudomonasaeruginosa, Eur J Respir Dis. 1985 August; 67(2):118-27.) shows that theinjection of ibuprofen sodium is able to reduce the inflammatory abilitycaused by pulmonary infection by P aeruginosa, but it does not affectthe biological activity of bacteria.

A recent work Juan D Guzman et al. (BMJ open Jun. 5, 2015“antitubercular specific activity of ibuprofen and the other2-arylpropanoic acids using the HT-spoti whole-cell phenotypic essay)shows that a formulation of ibuprofen and other anti-inflammatorynon-steroidal drugs present bactericidal properties for treatment oftuberculosis. Using a test called HT-SPOTi they demonstrate thatibuprofen is antibiotic, but an even more active derivative3,5-dinitro-Ibuprofen appears.

Another paper published by the group of Dr. Ahmed Mohsen (AntibacterialActivity of Anti-biofilm Some Non-steroidal Anti-Inflammatory Drugs andN-acetyl Cysteine against Biofilm Producing Some Uropathogens—AmericanJournal of Epidemiology and Infectious Disease, 2015, Vol. 3, No. 1,1-9) shows that drugs like NSAIDs and N-acetyl cystein adversely affectthe adherence of bacteria Staphylococcus aureus, Klebsiella pneumoniae,Proteus mirabilis and P. aeruginosa to the surface of catheters forurological use, which avoids undesired biofilms, and also present asignificant bactericidal effect, which can be very beneficial for thetreatment of infections caused by the use of catheters.

Research done by Dr. Cidalia Pina-Vaz et al. (Antifungal activity ofibuprofen alone and in combination with fluconazole against Candidaspecies Journal Medical Microbiology—Vol 49 (2000) 831-840), it is shownthat ibuprofen, alone or in combination with fluconazole, may cause asignificant antifungal effect on different strains of Candida, affectingthe stability of the membrane so as to produce a marked release ofintracellular K affecting the viability of the fungus.

Another patent, US20120115897 A1, describes the formation of a complexbetween ibuprofen and derivatives of esters of ascorbic acid and showsthat this formulation can enhance water solubility, facilitatingintravenous administration reducing application time, thereby loweringits undesired effect at the gastrointestinal level, increasing itspenetration into the blood-brain barrier and it can be applied invarious diseases such as arthritis, multiple sclerosis, cystic fibrosis,and pneumonia in the treatment of patent ductus arteriosus in prematureinfants, problems of cerebral hypoxia and certain cancers.

A common practice in the art is preparing ibuprofen in methanol, whichallows precipitation in the dilution of the solutions. Also, this NSAIDis commonly used in injectable form, which results in two problems. Thefirst is that sodium ibuprofen has a strong hemolytic effect, and secondis that ibuprofen rapidly interacts with albumin, the major plasmaprotein that when forming a complex strongly inhibits its bactericidalactivity as it is demonstrated in the studies herein, where it isclearly shown that, depending on the concentration of albumin present inthe medium, the bactericidal activity of ibuprofen decreases.

In addition, US 20130178448 A1 discloses a formulation of oraladministration of ibuprofen which is in the presence of different typesof lipids such as triglycerides or other fatty acids and alcohol, whichcan be administered as a liquid or tablets to be ingested orally byanimals so that they may reach high levels of ibuprofen in blood in thetreatment of lung diseases. It is herein demonstrated that whenibuprofen is solubilised in a triglyceride emulsion, as it happens inits interaction with albumin, it completely loses its bactericidalactivity.

As previously mentioned, when ibuprofen is solubilised in water itacquires surfactant properties. Therefore, it can interact with lipidmembranes affecting its stability. The interaction between ibuprofen andlipids and also its toxicity depends on the aggregation state ofibuprofen. It has been mentioned that at concentrations above its CMC,ibuprofen can damage the integrity of lipid membranes.

On the other hand, there is a history of the use of saline solution forthe treatment of airway disorders by nebulisation. Particularly, for thetreatment of cystic fibrosis nebulisation with antibiotics is used,being tobramycin the most common one. Furthermore, there is a history ofthe use of ibuprofen as an anti-inflammatory drug for the sameconditions, but usually by oral or intravenous administration. It isworth highlighting that patients with cystic fibrosis, in generalchildren, the continuous use of NSAIDs causes adverse effects that oftenworsen the pathological condition. In addition, as detailed herein thereis history suggesting the use of ibuprofen as a bactericidal drug,although the prior art suggests that it would not be suitable for useagainst P. aeruginosa and against Burkhordelia cepacia. Finally, anarticle in Journal of Aerosol Medicine and Pulmonary Drug Delivery inMarch 2009: “Analgesic Effect from Ibuprofen Nanoparticles Inhaled byMale Mice” discloses the nebulisation of solid ibuprofen carried insteam to rats, to assess its anti-inflammatory effect and demonstratethat it requires 3 to 5 times less concentration to have an effectequivalent to that in oral doses.

The present invention provides a composition that can be administered bynebulisation to treat cystic fibrosis, which presents an unexpected andsurprising effect by acting as a bactericide for type Gram+ and Gram−bacteria, especially by inhibiting bacteria such as P. aeruginosa, S.aureus, B cepacia and also having virucidal effect on thoselipid-enveloped viruses. The composition of the present invention isvery simple and its side effects are negligible, compared with thecontinuous use of common antibiotics for the treatment of this disease.One of the technical effects of the present invention is that thebactericidal effect of ibuprofen increased 5 times for being inhypertonic NaCl solution, as it can be seen in Example 11 herein.Moreover, providing the examples herein, the inventors have shown theability of ibuprofen above and especially below its critical micelleconcentration (CMC), to destabilize membranes of different Gram+ andGram− bacteria and lipid-enveloped viruses, affecting their biologicalactivity, allowing redefining, thanks to the present invention,ibuprofen as a bactericidal and virucidal agent, in combination with asaline solution.

BRIEF DESCRIPTION OF THE INVENTION

The bactericidal and virucidal pharmaceutical composition, to be appliedto epithelial tissue, such as lung, nose and mouth, the main object ofthe present invention, comprises an anti-inflammatory drug (NSAID) in aconcentration between 5 and 500 mM solubilised in saline solution,preferably hypertonic comprising a concentration of between 0.3 and 2Molar NaCl can be CIK; more preferably between 0.4 and 1, 1 Molar NaCl;even more preferably between 0.9 and 1, 05 Molar NaCl. Itsadministration form is selected from the group comprised by inhalation,nebulisation, mouthwash and topical administration. Wherein saidanti-inflammatory drug (NSAID), preferably comprises a concentrationbetween 5 and 180 mM; more preferably less than 180 mM; even morepreferably between 5 and 50 mM. Wherein said non-steroidalanti-inflammatory is arylpropionic selected from the group comprised byibuprofen, naproxen, flurbiprofen, ketoprofen, diclofenac, diflunisal,etodolac, fenoprofen, indomethacin, meclofenamate, mefenamic acid,meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, tolmetin,celecoxib, acetylated salicylates, non-acetylated salicylates, andcombinations thereof; preferably ibuprofen. And where their counterionsare monovalent cations selected from the group comprised of sodium,potassium, lithium and combinations thereof. Furthermore, it saidcomposition comprises a pH in aqueous solution between 6.0 and 8.5;preferably between 7.0 and 8.0; more preferably between 7.5 and 7.9.

Furthermore, the present invention may be embodied either in liquid oras a powder or lyophilised by drying or lyophilisation respectively fromthe hypertonic aqueous solution of said pharmaceutical composition.

In a preferred embodiment the present invention further comprises ananesthetic agent selected from the group comprised by xylocaine,lidocaine, carticaine, mepivacaine and mixtures thereof.

The present invention is a pharmaceutical composition for the treatmentof cystic fibrosis in nebulised application to reach the lungs, as wellas for the treatment of viral infections Herpes simplex type, applied toaffected skin. It can also be used as a bactericidal mouthwash.

In a preferred form of implementing the present invention for thetreatment of cystic fibrosis said pharmaceutical composition is in theabsence of usual antibiotics for this disease. These usual antibioticsinclude among others, colistimethate sodium, tobramycin, ciprofloxacin,lysine, levofloxacin, ciprofloxacin aztreonam, fosfomycin, amphotericinB, vancomycin, gentamicin, ceftazidime, ampicillin and amikacin andmixtures thereof.

In another alternative to implement the present invention, saidcomposition further comprises said usual antibiotics.

The process of manufacturing said bactericidal and virucidalpharmaceutical composition to be applied on epithelial tissues, such aspulmonary, nasal and oral tissue, another object of the presentinvention comprises the following steps:

A. said NSAID is mixed with water and stirred to maintain the suspensionof the NSAID;

B. NaOH is added until a pH between 6.8 and 8.5 is reached by gentlestirring, so as to allow complete solubilisation of said NSAID to aconcentration of between 1 and 100 mg/mL; preferably allowed to stand atroom temperature for at least 12 h;

C. a hypertonic NaCl saline solution is added to the preparation in stepB., at a concentration of 0.3 to 2 M;

D. the preparation in step D. is filtered through 0.22-micron porefilter.

In a preferred embodiment of the present invention, said process furthercomprises the following step:

E. the filtered solution of step D. is lyophilised;

Finally, after step E it is possible to add the next step:

F. when applied either to be vehiculated to the lungs by nebuliser or tobe applied to the mouth as a mouthwash, the lyophilised composition ofstep E. is resuspended in water or in a 5% glucose solution.

The present invention provides a bactericidal and virucidalpharmaceutical composition to be applied on epithelial tissues, such aspulmonary, nasal and oral tissue, comprising a non-steroidalanti-inflammatory drug (NSAID) and a salt. Where it preferably comprisesa molar ratio of said NSAID and said salt of between 1:0.6 and 1:400,more preferably between 1:10 and 1:100 and wherein said salt is selectedfrom the group comprising sodium chloride, potassium and combinationsthereof. And preferably such composition comprises a pharmaceuticalformulation to be diluted in water in the absence of any othercomponent. And where preferably it comprises a pharmaceuticalformulation for the treatment of cystic fibrosis. And where preferablyit comprises a pharmaceutical formulation for the treatment of HerpesSimplex. And where preferably comprises a pharmaceutical formulation formouthwash.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Chemical structure of ibuprofen

FIG. 2: Chemical structure of tobramycin

FIG. 3: Results from Example 4: Bactericidal effectiveness of solutionsof SODIUM IBUPROFEN (SODIUM IBUPROFENATE or IBUPROFEN SODIUM SALT)IBU-Na and IBUPROFEN (PROTONATED IBUPROFEN) IBU-H in P. aeruginosa

FIG. 4: Results from Example 5: Bactericidal effectiveness of IBU-Nasolutions.

FIG. 5: Results from Example 6: Antimicrobial effect of IBU-Na on P.aeruginosa as a function of time.

FIG. 6: Results from Example 7: Antimicrobial effect of IBU-Na on P.aeruginosa at different pH values.

FIG. 7: Results from Example 8: Antimicrobial effect of IBU-Na ondifferent bacterial strains

FIG. 8: Results from Example 9: Antimicrobial effect of IBU-Na atvarious concentrations on three main bacterial strains that areassociated with Cystic Fibrosis (CF).

FIG. 9: Results from Example 10: Antimicrobial effect of naproxen on apopulation of Burkhordelia cepacia.

FIG. 10: Results from Example 11: Effect of ionic strength (NaCl) on aculture of P. aeruginosa.

FIG. 11: Results from Example 12: Effect of the composition of thepresent invention comprising IBU-Na with ionic strength (1M NaCl) on aculture of P. aeruginosa.

FIG. 12: Results from Example 13: Bactericidal effect of the compositionof the present invention, comprising a hypertonic solution of ibuprofenas a function of time.

FIG. 13: Results from Example 14: Effect of the composition of thepresent invention comprising IBU-Na with ionic strength on a culture ofP. aeruginosa varying treatment time (minutes).

FIG. 14: Results from Example 15: Effect of the composition of thepresent invention comprising ibuprofen (IBU-Na) with ionic strength (1MNaCl) on a culture of three bacterial strains.

FIG. 15: Results from Example 16: Effect of the presence of humanalbumin on the bactericidal activity of IBU-Na [20 m] in medium ionicstrength (1M NaCl).

FIG. 16: Results from Example 17: Effect of LIPOVENOS in the presence ofionic strength (1 M NaCl) over a culture of P. aeruginosa.

FIG. 17: Results from Example 19: Antimicrobial effect of tobramycin asa function of concentration and contact time on P. aeruginosa.

FIG. 18: Results from Example 20: Antimicrobial effect of tobramycin inthe presence of ionic strength on P. aeruginosa.

FIG. 19: Results from Example 21: Antimicrobial effect of IBU-Na andtobramycin in the presence of ionic strength on an artificial biofilm ofP. aeruginosa.

DETAILED DESCRIPTION OF THE INVENTION

The bactericidal pharmaceutical composition, which also has virucidaleffects, which is to be applied on epithelial tissues, such aspulmonary, nasal and oral tissue, the main object of the presentinvention comprises a non-steroidal anti-inflammatory drug (NSAID) and asalt; wherein said NSAID is in a concentration between 5 and 500 mM andsolubilised in saline solution of said salt. Wherein said salinesolution is preferably hypertonic comprising a concentration of between0.3 and 2 Molar of a salt that is preferably NaCl, but can be any saltsuitable for human consumption such as potassium chloride; morepreferably between 0.4 and 1, 1 Molar NaCl; even more preferably between0.9 and 1, 05 Molar NaCl. Its administration form is selected from thegroup comprised by inhalation, nebulization, mouthwash and topicaladministration. Wherein said non-steroidal anti-inflammatory drug(NSAID), preferably comprises a concentration between 5 and 180 mM; morepreferably less than 180 mM; even more preferably between 5 and 50 mM.Wherein said non-steroidal anti-inflammatory agent is selected from thegroup comprised by ibuprofen, naproxen, flurbiprofen, ketoprofen,diclofenac, diflunisal, etodolac, fenoprofen, indomethacin,meclofenamate, mefenamic acid, meloxicam, nabumetone, oxaprozin,piroxicam, sulindac, tolmetin, celecoxib, acetylated salicylates,non-acetylated salicylates, and combinations thereof; preferablyibuprofen. And where their counterions are monovalent cations selectedfrom the group comprised of sodium, potassium, lithium and combinationsthereof. Furthermore, said composition comprises a pH in aqueoussolution between 6.0 and 8.5; preferably between 7.0 and 8.0; morepreferably between 7.5 and 7.9.

In a preferred embodiment of the present invention said pharmaceuticalcomposition comprises ibuprofen or sodium ibuprofen in a concentrationof 50 mM in a hypertonic saline solution 1 M of NaCl.

Furthermore, the present invention may be prepared either in liquidstate or as a powder or lyophilised by drying or lyophilisation,respectively from the hypertonic aqueous solution of said pharmaceuticalcomposition. Both the drying process and the lyophilisation ofpharmaceutical compositions is well known in the prior art, thereforeproviding further details on the subject is not considered necessary.

In a preferred embodiment, the present invention further comprises ananesthetic agent. The anesthetic effects of the composition of thepresent invention soothe the pain and discomfort caused by cysticfibrosis, but it is mainly beneficial when used for the treatment ofviruses such as Herpes. Such anesthetic agent can be any of the acceptedones for pharmaceutical use.

Among those anesthetics preferred for the present invention, thefollowing can be mentioned: xylocaine, lidocaine, carticaine,mepivacaine and the mixtures thereof.

The present invention is a pharmaceutical composition which is usefulfor the treatment of cystic fibrosis in nebulised application to reachthe lungs. In this really serious disease affecting mainly children, thepresent invention has advantages that had never been reported before,since with very low doses of NSAIDs not only anti-inflammatory but alsobactericidal and virucidal effects are achieved, without generatingresistant mucus and acting even if it already exists. The presentinvention is also applicable in the treatment of Herpes simplex typeviral infections, applied to affected skin. The present invention isalso effective as bactericidal oral rinse. The combination of ionicstrength with the presence of NSAIDs at very low doses achieves abactericidal and virucidal effect that has never been reported before.Technically, using very low concentrations of NSAIDs results in adramatic reduction of the common adverse effects of theseanti-inflammatory drugs.

In a preferred embodiment of the present invention to treat cysticfibrosis said pharmaceutical composition is free from the usualantibiotics for this disease. Such usual antibiotics are, among others,colistimethate sodium, tobramycin, ciprofloxacin, lysine, levofloxacin,ciprofloxacin aztreonam, fosfomycin, amphotericin B, vancomycin,gentamicin, ceftazidime, ampicillin and amikacin and the mixturesthereof.

In another alternative embodiment of the present invention, saidcomposition further comprises said usual antibiotics.

The process of manufacturing said bactericidal and virucidalpharmaceutical composition for application on lung, nose and mouthepithelial tissues, another object of the present invention comprisesthe following steps:

A. said NSAID is mixed with water and stirred to maintain suspension ofthe NSAID;

B. NaHO is added until a pH between 6.8 and 8.5 is reached by gentlestirring, so as to allow complete solubilisation of said NSAID to aconcentration of between 1 and 100 mg/mL;

C. It is allowed to stand at room temperature for at least 12 hours;

D. a hypertonic NaCl saline solution is added to the preparation in stepC., at a concentration of 0.2 to 2 M;

E. the preparation is filtered through 0.22-micron pore filter.

In a preferred embodiment of the present invention, said process furthercomprises the following step:

F. the filtered solution of step E. in claim 11 is lyophilised;

Finally, after step F it is possible to add the next step:

G. when applied either to be vehiculated to reach the lungs by means ofa nebuliser or to be applied to the mouth as a mouthwash, thelyophilised composition of step f. is resuspended in distilled water orin a 5% glucose solution.

Tests to Characterize the Bactericidal Properties of NSAIDs and of theComposition of the Present Invention.

There are numerous and varied laboratory methods that can be used todetermine in vitro the susceptibility of bacteria to variousantimicrobial agents such as plaque assays of inhibitory growth halos,growth/inhibition test in nutrient broths, commercial kits,antibiograms, etc.

In this case, tests were run on a non-nutritive liquid carrier (buffersolution) wherein the antimicrobial agent is mixed with quantifiedinocula of pure bacterial cultures. After different incubation times,the material is seeded on agar plates to perform quantification assayson the number of bacterias surviving the treatment.

The examples detailed below describe an overview of the tests, andcontains among others, the preliminary activities of conditioning ofstrains, work supplies, materials and methods and analysis of theresults obtained from biological activity.

Tests to Characterize the Virucidal Properties of NSAIDs and of theComposition of the Present Invention.

For the assessment of virucidal activity of NSAIDs, preferablyibuprofen, tests were conducted as described in Alasino et al in 2007.In this case, the various strains of virus with lipid envelope areincubated with increasing amounts of ibuprofen for a period rangingbetween 60 and 120 minutes. This material was then incubated withcultured cells which are susceptible to attack by these viruses, showinga marked cytopathic effect (cell aggregates (rosettes) or cell death)that can be detected by observation under the optical microscope.

Potential Applications of the Present Invention to Various InfectiousDiseases

The present invention provides a composition of ibuprofen sodium aloneor associated with a hypertonic saline pH between 6.8 and 8.2, havingbactericidal properties against different bacteria such as Pseudomonasaeruginosa, Burkhordelia cepacia, Pseudomonas fluorescens, Enterobacteraerogenes, Klebsiella pneumoniae, Escherichia coli, Bacillus subtilis(Gram+), Staphylococcus aureus (Gram+), Enterococcus faecalis (Gram+)and virucidal properties which produce the inactivation of thoselipid-enveloped viruses such as, bovine diarrhea virus, Herpes types Iand II, vesicular stomatitis virus, hepatitis C virus, varicella virus.

Applications of the composition of the present invention withbactericidal and virucidal properties may be used in the treatment ofinfectious diseases of the bacterial type that directly affect mucosasuch as oral or pulmonary mucosa, or else viral diseases as infectionsby Herpes type I, on lips, mouth or vaginal level, as well as in thecase of the cutaneous expression of Herpes virus type II.

Advantages of the Composition of the Present Invention in TreatingVarious Infections

The bactericidal properties of an IBU solution alone, preferably incombination with a hypertonic solution, is beneficial to treat bacterialinfections, especially those at the lung level caused by bacteria suchas P aeruginosa, which is capable of producing biofilms, as in the caseof cystic fibrosis, a disease that is very difficult to cure. Thecomposition of the present invention can be in liquid form, which makesit likely to be applied by a standard nebulisation system. Nebulisersare the devices of choice for pulmonary administration of drugs. Currentnebulisers work by ultrasonic pulses applied to a solution meant to forma cloud where the drugs to be vehiculated are found. Patients breath inthis cloud normally for several minutes until the solution is exhausted,normally 1 to 5 mL by nebulisation, which has some advantages asfollows:

-   -   In this case the drug would reach the lungs directly, rendering        concentration in the target organ more effective in order to act        on the bacteria P. aeruginosa, S aureus, methillicin resistant        and B cepacia as detailed in the examples mentioned below.    -   This would avoid the systemic use of antibiotics and NSAIDs        because when entering blood, the dilution affecting the        effective concentration in the target organ occurs.    -   It prevents the NSAID coming into contact with plasma components        and more specifically with the protein albumin with which it        interacts strongly and could reduce its bactericidal activity.    -   Considering that IBU is a molecule of anionic nature, it does        not present interaction with strongly anionic biopolymers such        as alginate and DNA, major materials present in the biofilm that        appears in this pathology, as it can occur with antibiotics        currently used such as tobramycin (FIG. 2) having five primary        amino groups in its structure.    -   The fact that the present invention comprises ibuprofen together        with a solution of high ionic strength, allows as it is shown        herein, increasing effectiveness of bactericidal power 5 times,        reducing the contact time to develop its bactericidal property        from hours to a few minutes.    -   The composition of the present invention, while presenting a        hypertonic saline solution allows it to act not only as a        biofilm fluidizing agent, but also presents a sound bactericidal        effect, in addition to its anti-inflammatory effect already        known.

In the particular case of the bactericidal effect of the composition ofthe present invention on the oral epithelium, the advantage of thisformulation against current antiseptic formulations lies in that thosewhich use chlorhexidine eventually cause teeth coloring, while this doesnot happen with the use of ibuprofen. Moreover, the composition of theinvention can be used for buccal or labial infections caused by HerpesSimplex Type I viruses since in addition to its antiviral effect itprovides anti-inflammatory effect during treatment, a desired conditionto reliever discomfort during infection.

Manufacturing Process of the Composition of the Present Invention

Said composition of the invention comprises at least one bioactive agentsuch as NSAIDs solubilised in aqueous medium in addition to theclassical anti-inflammatory property, in the present invention exhibitsbactericidal and virucidal properties when said NSAID is formulated at aconcentration below its critical micelle concentration and in thepresence of saline solution. The manufacturing process of saidcomposition comprises for instance, the following steps:

-   -   NSAID is placed in a container into which water is added; it is        then stirred to maintain NSAIDs in suspension,    -   a basic solution such as NAHO is subsequently added to reach a        pH between 7.0 and 8.5 stirring gently, so as to allow complete        solubilisation of said NSAID. Preferably, it is allowed to rest        at room temperature for at least 12 hours,    -   the preparation is diluted to reach the desired concentration of        NSAID for the treatment chosen,    -   a NaCl saline solution is added to the previous preparation,        comprised within a 0.2 to 2 M range,    -   the preparation is passed through a 0.22-micron pore size        filter,    -   the sterilized material can be finally lyophilised for        preservation or else kept in liquid state until it is used.    -   Finally, at the time of application, either to be vehiculated to        the lungs by means of a nebuliser, or to be applied to the mouth        as a mouthwash, the lyophilised NSAID is resuspended in the        original volume of: a—distilled water, b—in a 5% glucose        solution.

Said NSAIDs may be selected from the group comprised by arylpropionicselected from the group comprised by ibuprofen, naproxen, flurbiprofen,ketoprofen, diclofenac, diflunisal, etodolac, fenoprofen, indomethacin,meclofenamate, mefenamic acid, meloxicam, nabumetone, oxaprozin,piroxicam, sulindac, tolmetin, celecoxib, acetylated salicylates,non-acetylated salicylates, and combinations thereof.

Salts of said NSAIDs such as Na, I or K are preferred. Therefore, whenadding sodium hydroxide solution is added, NSAID salt is formed.Preferably, sodium ibuprofen (IBU-Na) is used, for it presents greatersolubility in aqueous solution than protonated ibuprofen (IBU-H).

The composition of the present invention comprises powder NSAID,preferably sodium ibuprofen, and Na or K salt in a molar ratio ofNSAID:salt of 1:0.6 and 1:400, preferably between 1:10 and 1:100.

This powder may be diluted in water to obtain the pharmaceuticalformulation of the invention.

In this detailed description, IBUPROFEN means ibuprofen in any of itsforms. In particular, PROTONATED IBUPROFEN is to be identified with thefollowing name:

IBU-H.

While SODIUM IBUPROFEN, also known as SODIUM IBUPROFENATE or IBUPROFENSODIUM SALT shall be identified as IBU-Na herein. This sodium ibuprofencan be free of water in mono hydrate or dihydrate form.

EXAMPLES Example 1

Preparation of Solutions for Tests Performed Herein and in the FollowingExamples.

Next, methods of preparation of the components used for the embodimentof the present invention, which prove to be the best known to theinventors are described, but they are not the only possible ones.

Preparation of IBU-H Solutions:

Solid ibuprofen is dissolved (20 g in pure ethanol up to 100 mL); thisrepresents IBU-H 1M solution. Studies on the activity of theseformulations use a solution that placed on the culture medium does notexceed 5% ethanol concentration which does not affect the viability ofbacteria.

IBU-Na Solution Preparation:

IBU-Na stock solution is prepared by weighing 20 g of ibuprofen, addingwater to 50 mL, and then adding enough NAHO 4M solution to reach a pHbetween 7 and 8; finally it is made up to 100 mL to a finalconcentration of 1 M Na-IBU.

Naproxen Solution Preparation:

Naproxen stock solution is prepared by weighing 23 g of Naproxen, addingwater to 50 mL, and then adding enough NAHO 4M solution to achieve a pHbetween 7 and 8; finally it is made up to 100 mL to reach a finalconcentration of 1 M Naproxen.

Preparation of the Favorite Composition of the Present Invention:

-   -   The following examples show the steps followed to obtain the        various solutions used:        -   a. 2 g of ibuprofen are placed in an Erlenmeyer that is            added with 5 mL of water and stirred to maintain the            ibuprofen suspension,        -   b. then NAHO 4 M is added to reach a pH comprised between            7.0 to 8.5 stirring gently, so as to allow complete            solubilisation of IBU and it is finally made up to 10 mL to            achieve a concentration of 200 mg/mL. It is allowed to stand            at room temperature for at least 12 hours.        -   c. The preparation is diluted to reach the desired IBU-NA            concentration for the treatment chosen, as in the example.        -   d. The hypertonic saline solution is added to the            preparation mentioned above to reach a concentration of 1 M            NaCl.        -   e. The preparation is filtered through a 0.22-micron pore            filter.

To obtain the composition of the present invention for use in the testsperformed in the following examples, compositions ranging from 1 to 100mM of IBU-Na and hypertonic solutions, which present a finalconcentration of 1 M NaCl, are obtained following the proceduredescribed. The procedures followed are the usual laboratory practices toperform dilutions.

Example 2

Activation and Conservation of Bacterial Strains Stock

Lyophilised commercial strains ATCC (KWIK-STIK MicroBioLogics) activatedin tubes containing 10 mL of Brain Heart Infusion BHI (Biokar BK015HA)at 37° C. for 24 hours were used.

Composition in g/L:

Brain and heart Infusion 17.5 Peptone 10 Glucose 2 Sodium chloride 5Disodium phosphate 2.5

Preparation: 37 g of medium formulated in 1 L of distilled water weredissolved, heated until completely dissolved and adjusted in pH to7.4±0.2 at 25° C.; finally, it is autoclaved for sterilization for 15minutes at 121° C.

After the activation time of each strain in said means and havingobserved effective growth by turbidity close to 0.5 scale Mc Farland'sstandard, 10% v/v sterile glycerol as cryoprotectant was added to eachculture. Subsequently, the content of each tube was distributed in 1 mlaliquots in 1.5 mL eppendorf tubes. These strains were identified asstock strains and stored at ultra low temperature in a freezer at −70(Sanyo MDFU70V Ultra low temperature freezer) to provide an adequatereserve of each stock strain.

Example 3

Antimicrobial Activity Tests of IBU

IBU effectiveness testing at different concentrations, times of action,ionic strength, pH variations and various bacterial populations wereperformed; effective concentrations of commercial tobramycin antibiotic(FIG. 2) were also measured.

Test Methodology

Work consisted of incubating bacterial populations of a known load interms of CFU/mL with the compounds mentioned in different molarconcentrations. In vitro test batteries were organized at differentdoses or concentrations and in 1.5 mL Eppendorf tubes without theaddition of culture medium. To this purpose, a PBS 100 mM phosphatebuffer was used to maintain physiological conditions throughout theperiod of each test at 37° C.

PBS 100 mM Buffer Composition:

8 g (137 mM) NaCl 0.2 g (2.7 mM) KCl 1.44 g (10 mM) NaHPO 0.24 g (2 mM)KH2PO 1000 mL Milli Q water

When the time for each assay finished, aliquots of 100 uL and decimaldilutions of each tube which were seeded on petri dishes for the directcount of colony forming units (CFU) in Mueller Hinton agar (MHA), usingpour-plating or spread-plating standard methods.

Each plate was added with 5 uL of a solution of 2,3,5-metabolic dyetriphenyltetrazolium of chloride (TTC) 1% to enhance the contrast ofcolonies. Plates were incubated in inverted position, in a culture stovefor 24-48 hours at 37° C. and subsequently the direct count was done,adjusted by dilution factors and seeding to report results in terms ofCFU/mL.

The medium composition in g/L is:

Peptone 17.5 Starch 1.5 Meat extract 4 Agar 15

Preparation:

22.5 g of the formulated product are dissolved in 1 L of distilled waterand boiled. pH is adjusted to 7.3±0.2 at 25° C. and autoclaved for 15minutes at 121° C.

During testing, the medium was stored in thermostatic bath between45-50° C. because the seeding technique requires imbibing andintegrating bacteria in the medium. Once solidified, the plates wereincubated in inverted position for a set time of 24-48 hours prior toeach count for the purposes of assessing the effective concentrations ineach case and comparing with other treatments.

Example 4

Bactericidal effectiveness of IBU Na and IBU-H solutions in P.aeruginosa The test results are shown with IBU-Na and IBU-H solutions atdifferent molar increasing concentrations on a population of P.aeruginosa at an incubation time of 4 h at 37° C. and subsequentseeding.

To perform the test of bactericidal effectiveness of the solution ofIBU-H a final concentration of ethanol EtOH <5% was ensured to preventthe bactericidal action of the solvent itself.

IBU P. aeruginosa (UFC/mL ± 10) [mM] IBU-Na IBU-H. 0 (Control) 1,700,0001,700,000 1 1,700,000 1,700,000 5 1,700,000 1,700,000 10 760,000 480,00020 300,000 200,000 50 50 1000 100 10 10

See FIG. 3, where the results of this test shown that IBU-Na has ahigher bactericidal capacity than IBU-H.

Example 5

Bactericidal Effectiveness of IBU-Na Solutions

In this example, the results shown correspond to a test with IBU-Nasolutions at different increasing molar concentrations on threepopulations of the same strain (P. aeruginosa) for an incubation time of4 h at 37° C. and subsequent plating.

IBU-Na P. aeruginosa (UFC/mL ± 10) [mM] Population A Population BPopulation C 0 (Control) 200,000 20,000 2,000 1 200,000 20,000 2,000 5150,000 17,000 1,800 10 100,000 3,500 800 20 20,000 400 50 30 500 40 1040 100 10 10 50 10 10 10 100 10 10 10

As shown in FIG. 4, this study concludes that the effective dose ofIBU-Na depends on the bacterial load.

Example 6

Antimicrobial Effect of IBU-Na on P. Aeruginosa as a Function of Time.

The effect of IBU-Na on the same strain of P. aeruginosa at a fixedconcentration of bacteria varying the concentration and contact time wascharacterized. The test was performed at 37° C. and subsequently thesamples were plated.

P. aeruginosa count (UFC/mL ± 10) IBU-Na As a function of treatment time[mM] 1 hour 4 hours Overnight (O.N.) 0 (Control) 2,000,000 2,000,0002,000,000 1 2,000,000 2,000,000 2,000,000 2.5 2,000,000 1,800,0002,000,000 5 1,700,000 1,300,000 1,000,000 10 1,000,000 1,000,000 900,00025 500,000 250,000 100,000 50 100,000 30 10 100 2,000 10 10

As it can be seen in FIG. 5, these results show that certain IBU Nacontact or treatment time is required to obtain antimicrobial effects;it was standardized for most trials in 4 hours.

Example 7

Antimicrobial Effect of IBU-Na on P. aeruginosa at Different pH Values.

Study on the effect of pH of the incubation medium test on bactericidalpotential of a 50 mM solution of NaIBU incubated for 4 h at 37° with aninoculum of P. aeruginosa

IBU-Na P. aeruginosa (UFC/mL ± 10) [mM] pH 6.8 pH 7.3 pH 7.8 0 (Control)200,000 200,000 200,000 50 10 10 600

As it can be seen in FIG. 6, the result shows that IBU-Na acts as anantimicrobial agent in the range of physiological pH values.

Example 8

Antimicrobial Effect of IBU-Na on Different Bacterial Strains

Antimicrobial effect of IBU-Na at two test concentrations [50 and 100mM] on the viability of Gram positive and negative bacterial strainsduring 4-hour contact at 37° C.

Colony count (UFC/mL ± 10) IBU-Na IBU-Na Microorganism Controls 50 mM100 mM A Pseudomonas aeruginosa 2,000,000 100 10 B Burkhordelia cepacia1,300,000 680 10 C Pseudomonas fluorescens 100,000 150 10 D Enterobacteraerogenes 1,000,000 450 10 E Klebsiella pneumoniae 1,000,000 600 10 FEscherichia coli 1,000,000 500 10 G Bacillus subtilis (Gram +) 500,000250 10 H Staphylococcus aureus (Gram +) 1,000,000 550 10 I Enterococcusfaecalis (Gram +) 100,000 180 10

As it can be seen in FIG. 7, the result shows that IBU-Na acts anantimicrobial agent on different bacterial strains (Gram positive andnegative ones).

Example 9

Antimicrobial Effect of IBU-Na at Various Concentrations on Three MainBacterial Strains Associated with Cystic Fibrosis (CF).

Effect of IBU-Na at various concentrations on the viability ofStaphylococcus aureus, Burkhordelia cepacia and Pseudomonas aeruginosa,for 4 hours at 37° C.

IBU-Na Colony count (UFC/mL ± 10) [mM] Staphylococcus BurkhordeliaPseudomonas 0 (Control) 1,000,000 1,300,000. 1,700,000.  5 1,000,0001,300,000 1,700,000 10 1,000,000 1,300,000 760,000 25 30,000 40,000200,000 50 600 700 100 100  10 10 10

The result shows that IBU-Na has effect on the three most commonbacterial strains which are found in cystic fibrosis (FIG. 8)

Example 10

Antimicrobial Effect of Naproxen on a Population of Burkhordeliacepacia.

In this study, the effect of naproxen on a population of B. cepaciastrain was analyzed at different concentrations, testing for 4 hincubation at 37° C.

B. cepacia Naproxen [mM] (UFC/mL ± 10) 0 (Control) 130,000 10 125,000 2538,000 50 31,000 100  1,300

As shown in FIG. 9. The result shows that naproxen shows lessantimicrobial activity compared with IBU-Na.

Example 11

Effect of Ionic Strength (NaCl) on a Culture of P. aeruginosa.

In this study the effect of the ionic strength (NaCl) on a culture of P.aeruginosa is analyzed at different concentrations in treatments lasting4 hours at 37° C.

P. aeruginosa NaCl [M] (UFC/mL ± 10) 0 (Control) 2,000,000 0.012,000,000 0.025 2,000,000 0.05 2,000,000 0.1 2,000,000 0.25 1,800,0000.5 1,600,000 1 1,000,000

No decrease was observed in the bacterial population as a result of theeffects of ionic strength. (FIG. 10)

Example 12

Effect of the Composition of the Present Invention Comprising IBU-Nawith Ionic Strength (1M NaCl) on a Culture of P. aeruginosa.

Study to evaluate the bactericidal effect in the presence IBU-Na ionicstrength (1 M NaCl) on a P. aeruginosa culture for 4 hours at 37° C.

P. aeruginosa (UFC/mL ± 10) IBU-Na ionic strength Without ionic [mM]NaCl (1M) strength 0 (Control) 1,700,000 1,700,000  5 1,000 1,700,000 1010 760,000 25 10 200,000 50 10 100

The result clearly shows that ionic strength enhances the action of theIBU-Na, reducing the needed dose to obtain antimicrobial effects on thepopulation of bacteria tested. FIG. 11 shows clearly that with aconcentration of 25 mM of IBU-Na the number of CFU/mL decreases to 1000,while the composition of the present invention with ionic strength ofNaCl to 1 M the same result is obtained at a concentration five timeslower, i.e., about 5 mM Na-IBU.

Example 13

Bactericidal Effect of the Composition of the Present Invention,Comprising a Hypertonic Solution of Ibuprofen as a Function of Time.

Following, the study to evaluate the bactericidal effect IBU-Na in thepresence of ionic strength (1M NaCl) at different times of incubation ona culture of P. aeruginosa is shown. The treatments lasted 1 hour, 2hours and 4 hours at 37° C. pH 6.5.

P. aeruginosa IBU-Na [mM] + (UFC/mL ± 10) ClNa (1M) 1 hour 2 hours 4hours 0 (Control) 800,000 800,000 800,000  5 16,000 13,000 8,500 10 3030 10 20 10 10 10

As seen in FIG. 12, this result shows that ion strength can shortenaction times previously set.

Example 14

Effect of the Composition of the Present Invention Comprising IBU-Nawith Ionic Strength on a Culture of P. aeruginosa Varying Treatment Time(in Minutes).

This study shows the bactericidal effect of IBU-Na 20 mM in the presenceof ionic strength (1M NaCl) on a culture of P. aeruginosa. Treatments atdifferent incubation times (1, 3 and 10 minutes)

P. aeruginosa (UFC/mL ± 10) IBU Preincubation 10′ with Time 20 mM withIBU 20 mM without ionic strength (Minutes) ionic strength ionic strengthPosterior: IBU 20 mM 0 1,000,000 1,000,000 1,000,000 (Control) 1 101,000,000 1,000,000 3 10 1,000,000 1,000,000 10  10 1,000,000 1,000,000

As seen in FIG. 13, the result shows that only the combined treatment ofibuprofen in the presence of ionic strength (IBU-Na 20 mM+NaCl 1 M)allows a synergistic result on the antimicrobial effect as a function oftime.

Analyzing other alternatives, as shown in the third and fourth column ofthe table in this same example with ibuprofen alone without ionicstrength and a dissociated treatment preincubating an inoculum of P.aeruginosa 1×10 8 CFU/mL for 10 minutes CINa 1 M and subsequent washingby dilution 1/10 in buffer containing 20 mM IBU. As a result it isstated that just ionic strength alone does not affect the inoculum; theionic strength pretreatment and subsequent treatment with ibuprofen didnot cause changes in the inoculum compared with the controls, as shownin the fourth column of the table in this example. As evidenced in thisexample, the present invention has an unexpected and surprisingtechnical effect, by achieving an almost instant synergistic effect (inrelation to other therapeutic options) that promotes the interaction ofibuprofen on microorganisms, and thus in just one minute achieving adramatic decrease of the population of microorganisms typical of cysticfibrosis.

Example 15

Effect of the Composition of the Present Invention Comprising Ibuprofen(IBU-Na) with Ionic Strength (1M NaCl) on a Culture of Three BacterialStrains.

Test to evaluate the effect of IBU-Na with ionic strength (1M NaCl) on 3bacterial strains: P aeruginosa, S aureus and B cepacia in 4-hourtreatments at 37° C.

IBU-Na [mM] + Colony count (UFC/mL ± 10) ClNa (1M) P. aeruginosa S.aureus B. cepacia 0 3,000,000 1,000,000 1,700,000 12.5 250 100 100 25 1010 10 50 10 10 10

As seen in FIG. 14, the presence of a hypertonic solution favors theantibacterial action of IBU-Na on the three strains of interestassociated with CF.

Next, application examples are given to show how the bactericidal effectof ibuprofen is affected when administered orally or intravenously.These examples show aspects that evidence the high inventive step of thepresent composition, since it provides a formulation, which avoidsfactors that have usually hidden the bactericidal effects of ibuprofen.

Example 16

Effect of the Presence of Human Albumin on the Bactericidal Activity ofIBU-Na [20 mM] in Medium with Ionic Strength (1M NaCl).

Study to evaluate the effect of the presence of a serum protein, humanalbumin, known for its ability to interact strongly with the ibuprofenmolecule on the bactericidal activity. In this case the initialpre-incubation was performed at room temperature for 1 hour, withdifferent concentrations of Albumin IBU-Na [20 mM]; they weresubsequently incubated with the bacteria in the presence of ionicstrength (1 M NaCl) to start the final treatment lasting 4 hours at 37°C.

Albumin (mg/mL) with P. aeruginosa IBU 20 mM y NaCl (1M) (UFC/mL ± 10)Control* 1,000,000 10  1,000,000 5 1500   2.5 10 *Albumin 10 mg/mLwithout IBU-Na

As it can be seen in FIG. 15, the presence of albumin interferes withthe antibacterial action of 20 mM IBU-Na. From albumin concentrationsbelow 10 mg/mL the bactericidal effect of IBU-Na, starts recovering,which suggests some sort of interaction.

Example 17

Effect of LIPOVENOS™ (Medium Chain Triglycerides) in the Presence ofIonic Strength (1 M NaCl) on a Culture of P. aeruginosa.

An inoculum of P. aeruginosa was incubated in the presence ofLIPOVENOS™+ionic strength (1 M NaCl) during a 4-hour treatment at 37° C.

LIPOVENOS % treatment P. aeruginosa with NaCl (1M) (UFC/mL ± 10) Control(0%) 2,000,000 2.5%  2,000,000  5% 2,000,000 10% 2,000,000 20% 2,000,000

As it can be seen in FIG. 16, LIPOVENOS™ has no effect on the inoculumat the concentrations tested.

Example 18

Effect of Na IBU-[20 mM] in the Presence of LIPOVENOS™ on the Viabilityof P. aeruginosa.

LIPOVENOS initial preincubation at different concentrations with IBU-Na20 [mM] at room temperature for 1 hour. It was subsequently mixed withbacteria to begin a treatment of 4 hours at 37° C.

LIPOVENOS(%) + P. aeruginosa Ibu 20 mM (UFC/mL ± 10) Control* 1,000,000LIPO 10% + IBU 20 Mm 1,000,000 LIPO 5% + IBU 20 Mm 1,000,000 LIPO 2.5% +IBU 20 Mm 1,000,000 *LIPOVENOS^(MR) 10%, without IBU-Na

The presence of LIPOVENOS™ up to 2.5%, exhibits a marked interference onthe antibacterial action of IBU-Na 20 mM.

Example 19

Antimicrobial Effect of Tobramycin as a Function of Concentration andContact Time on P. aeruginosa.

This test evaluated the activity of an antibiotic known as tobramycin ona population of P. aeruginosa in treatments lasting 1 h, 4 h and O.N. at37° C. and thus compared studies of previous examples of ibuprofenactivity.

P. aeruginosa Tobramycin (UFC/mL ± 10) [mM] 1 hour 4 hours O.N. Control2,000,000 2,000,000 2,000,000 1 1,000,000 30,000 30,000   2.5 60,00020,000 10 5 30,000 1,000 10 10  1,000 10 10

As it can be seen in FIG. 17, as bacteria and tobramycin antibioticcontact time increases, its bactericidal effect is enhanced.

Example 20

Antimicrobial Effect of Tobramycin in the Presence of Ionic Strength onP. aeruginosa.

This example shows an assay to assess a formulation of tobramycinantibiotic in the presence of 1 M NaCl solution on a population of P.aeruginosa for 4 h at 37° C.

Tobramycin [mM] P. aeruginosa with ClNa (1M) (UFC/mL ± 10) 0 1,000,0000.25 400,000 1 10 5 10 10 10

As shown in FIG. 18, with the presence of ionic strength (1M NaCl) thebactericidal effect of antibiotic tobramycin is enhanced on a populationof P. aeruginosa in a contact time of 4 h at 37° C.

Example 21

Antimicrobial Effect of IBU-Na and Tobramycin in the Presence of IonicStrength on an Artificial Biofilm of P. aeruginosa.

Comparative study of the effect of IBU-Na and tobramycin in the presenceof ionic strength (1 M NaCl) on an artificial biofilm of P. aeruginosain the presence of 2% alginate solution. Treatments lasted 4 hours at37° C.

P. aeruginosa Treatments (UFC/mL ± 10) Biofilm Control 2,000,000Biofilm + Fl (1) 2,000,000 (1) + IBU-Na 10 mM 25,000 (1) + IBU-Na 25 mM200 (1) + Tobramycin 5 mM* 10 (1) + Tobramycin 10 mM* 10 *Formation ofinsoluble complex

As it can be seen in FIG. 19, the test shows that in the presence ofionic strength IBU-Na kept its antimicrobial properties despite thealginate barrier and did not form an insoluble complex. Whereastobramycin formed an insoluble complex during the tests, but retainedits effectiveness as an antibiotic.

It is worth highlighting that it is an insoluble compound whichgenerates a progressive obstruction of the lungs in cystic fibrosis;Therefore, it is to be expected that when developing trials in humansthe composition of the present invention is likely to constitute arecommended therapy for this disease.

Example 22

Evaluation of the Potential Cytotoxic Effect of Nebulised Ibuprofen inTwo Concentrations on Lung Tissue in Rats.

This histopathological study analyzed the potential cytotoxic effect ofibuprofen potential on lungs at concentrations of 25 and 50 mM. Groups Aand B correspond to replicated treatments with 8 animals at eachconcentration; these were nebulised 1 hour per day for 4 months. Theremaining groups, C and D are similar to A and B, but after 4 months ineach case they were allowed 15 days without any treatment and thensacrificed.

Doses Acute damage parameters Damage parameters sub-acute IBU AlveolarInterstitial Hyaline Proteinaceous septal Masson Granuloma andTreatments [mM] infiltration infiltration membranes material thickeningbodies giant cells A 25 0 1 1 1 1 NO NO Alveolar Hemorrhage 50 0 0 0 0 1NO NO Peribronchial Capillary Scarce and mononuclear congestion focalintense infiltrate B 25 0 1 0 0 1 NO NO 50 0 1 0 0 1 NO NO Peribronchialmononuclear intense infiltrate C 25 0 1 0 0 1 NO NO Peribonchiolar andperivascular mononuclear 50 0 1 0 0 1 NO NO Peribronchial mononuclearmild infiltrate D 25 0 1 0 0 1 NO NO Peribonchiolar and perivascular 500 1 0 0 1 NO NO Peribronchial mononuclear mild infiltrate

From the results of anatomopathological studies shown in the table, itcan be concluded that daily nebulisation of 60 minutes over 4 months,which is a relatively long period of time, a solution of ibuprofen 50 mMon Balb7c rat strains does not cause significant alterations onpulmonary tissue so as to render suspension necessary.

Example 23

Studies of Antiviral Activity of a Solution of Ibuprofen

There follows the description of in vitro studies where the activity ofa solution of sodium ibuprofen is shown to cause the inactivation ofviruses called “enveloped”, which are those viruses having a lipidenvelope.

Cell Culture Used in the Activity Assay

Kidney cells from African green monkey (Vero) (ATCC CCL-21), Madin Darbybovine kidney (MDBK) (ATCC CCL-22) cells and human cells from epitheliallarynx tumor (Hep 2) (ATCC CCL-23) were propagated in minimal essentialmedium (MEM) supplemented with 10% fetal bovine serum irradiatedcontaining 10000 IU of penicillin and 2 mM glutamine.

Viruses Used for Testing Inactivation

The BVDV bovine diarrhea virus was provided by INTA Castelar, and it waspropagated in MDBK cell monolayers. The vesicular stomatitis virus (VSV)(ATCC VR-158) and Herpes Simplex 1 virus (HSV) were propagated in VEROcells. Rubella virus strain (MV) was obtained from the Rouvax commercialvaccine (Paster Merieux. France) was propagated in Hep-2 cells. TheHerpes Simplex Virus (HSV) was provided by the Institute of Virology ofthe UNC, Universidad Nacional de Cordoba.

Virus Stock Preparation

Viruses were propagated using cells that are susceptible of infection,which were grown at 37° C., until complete destruction of the cellmonolayer. Cell-associated viruses were extracted using threefreeze-thaw cycles of the bottles that contained the cells. The materialwas centrifuged at 3000×g for 15 minutes and the supernatant wasfractionated and stored at −70° C. until use.

Tests to Evaluate the Virucidal Activity of Ibuprofen and of theComposition of the Present Invention.

Infectivity assays of HSV, BVDV, VSV and MV virus: infectivity virusactivity was determined by the titration method (by dilution 1/10) in96-well plates (NUNC Life Technologies, Rockville, Md., USA), applyingthe criterion of measuring the cytopathic effect on cells (aggregationand cell death) and therefore positive or negative is determined. 1/10serial dilutions of viruses were prepared in quadruplicate and incubatedfor 60 minutes at 37° C. stirring gently at different concentrations ofibuprofen ranging from 1 to 50 mM. After this period, the virus solutionwith ibuprofen is incubated in the presence of the cells and is alsoallowed to stand for 60 min. at 37° C. in order to allow the adsorptionof the virus on the cells so they can exert their cytopathic effect.Then 150 uL of maintenance medium is added to each well without removingthe inoculum.

Incubation was carried out at 37° C. in a stove containing 5% atmosphereof C02, 90% relative humidity for 3 days the case of HSV and VSV virusor up to 7 or 8 days for MV and BVDV virus, to allow sufficient time todetect the appearance of a potential cytopathic effect.

Determination of Infectious Dose 50

The so called Infectious Dose of cell culture 50% (TOD50 mL-1) wascalculated by the Reed-Muench method of analysis (Reed L I, H. Munch,Am. J. Hygiene 1938 27, 493.) for wells infected by positive viruses.

Test Results on the Effect of Ibuprofen at 50 mM Concentration on theInfectivity of Viruses.

Reduction of Virus viral level Characteristics Ibuprofen Virus FamilyGenome Size (nM) TCID50 mL−1 HSV Herpes dsDNA 120-200 >6.9 BVDV FlaviRssRNA 50-70 >6.5 VSV Rhabdo SssRNA  70-170 >7.7 MV Paramyxo SssRNA150-300 >5.0

As it can be seen from the results obtained, incubation of ibuprofen inthe presence of the enveloped viruses used, causes a marked inhibitoryeffect on the viral infectivity. Titers obtained expressed as InhibitoryDosage 50, range from 10⁵ and 10⁷ log CFU.

What is claimed is:
 1. A pharmaceutical composition comprising ibuprofenor a pharmaceutically acceptable salt thereof in a concentration from 5to 50 mM solubilized in a hypertonic saline solution comprising between0.3 M and 2 M sodium chloride.
 2. The composition of claim 1, whereinthe composition is in a form suitable for administration by inhalationor is in a form suitable for nebulization or is a mouthwash or is in aform suitable for topical administration.
 3. The composition of claim 1,wherein said composition comprises a pharmaceutically acceptable salt ofibuprofen selected from the sodium salt of ibuprofen, the potassium saltof ibuprofen, the lithium salt of ibuprofen and a combination thereof.4. The composition of claim 1, further comprising a pH in aqueoussolution of between 6.0 and 8.5.
 5. The composition of claim 1, furthercomprising an anesthetic agent.
 6. The composition of claim 5, whereinsaid anesthetic agent is selected from the group consisting ofxylocaine, lidocaine, mepivicaine and caricaine.
 7. The composition ofclaim 1, wherein said composition comprises a state selected from thegroup consisting of liquid, powder and lyophilized state.
 8. Thecomposition of claim 1, wherein said composition is in a form suitablefor nebulization.
 9. The composition of claim 1, further comprising anantibiotic selected from the group consisting of colistimethate sodium,tobramycin, ciprofloxacin, lysine, levofloxacin, ciprofloxacinaztreonam, fosfomycin, amphotericin B, vancomycin, gentamicin,ceftazidime, ampicillin, amikacin and mixtures thereof.
 10. Thecomposition of claim 1, wherein a molar ratio of said ibuprofen orpharmaceutically acceptable salt thereof to sodium chloride of thehypertonic solution is between 1:0.6 and 1:400.
 11. The pharmaceuticalcomposition of claim 1, wherein the composition comprises the sodiumsalt of ibuprofen.
 12. The pharmaceutical composition of claim 11,wherein the pH of the composition is between 6.0 and 8.5.
 13. Thepharmaceutical composition of claim 11, wherein the hypertonic salinesolution comprises between 0.4 M and 1 M sodium chloride.
 14. Thepharmaceutical composition of claim 13, wherein the concentration ofsodium ibuprofenate is 50 mM.
 15. The pharmaceutical composition ofclaim 11, wherein the concentration of sodium ibuprofenate is 50 mM. 16.The pharmaceutical composition of claim 1, wherein the hypertonic salinesolution comprises between 0.4 M and 1 M sodium chloride.